1. Field of the Invention
This invention relates generally to methods for the fabrication of surfaces punctuated by microscopic spikes or microcapillary structures. More specifically, this invention relates to techniques for forming surfaces of microscopic spikes or microcapillary structures by deposition of a material onto or through a track-etch membrane. Such structures have many applications, including cell transformation by microimpalement.
2. Description of the Prior Art
The ability to generate microscopic structures has had a major impact on many fields of science and technology, particularly in the electronics industry. Microfabrication is beginning to find applications in more diverse areas such as biotechnology as well. An example of a class of microfabricated structures is synthetic microtubules, which have been proposed to have applications in drug delivery systems, microwave components, electro-optical devices, vibration detectors, and anti-fouling paints for ships' hulls. See Pool, "101 uses for tiny tubules", Science 247:1410-1411 (1990). However, for many applications, the high cost of microfabricated structures currently prohibits their routine use. Novel fabrication methods will allow the cost of microfabricated structures to be reduced.
One application of microstructures that has been proposed recently is a novel method of introducing biomolecules into living cells, referred to as "microimpalement." This is described in my copending application, identified above. In that method, a bed of upright needles is provided onto which DNA or other biomolecules are loaded. Cells are forced onto the needles, after which an electric field is applied to cause the biomolecules to migrate into the cells' interior. The cells are then removed from the needles. The needles can comprise either simple spike structures, which can be coated with biomolecules, or they may comprise microcapillary structures, which may be filled with a solution of desired biomolecules. The width of each needle structure should be controlled such that it would be on the order of one-tenth of the diameter of the cells to be impaled, and the height should be about 2 to 5 microns. Thus, it becomes necessary to be able to manufacture inexpensive surface films or substrates with microstructures of defined dimensions.
Methods have been devised in the electronics industry for microfabrication of circuits and miniature devices. Using lithographic techniques, it is currently possible to produce devices with line widths down to 0.5 micron or less. However, in producing surfaces of microcapillaries with dimensions appropriate for microimpalement, it would be difficult to achieve the necessary aspect ratios (ratio of height to width). In addition, the cost of fabricating surfaces by techniques such as electron-beam lithography would make this method cost prohibitive. For applications such as microimpalement, where the precise dimensions and uniformity of the microstructures are not critical, methods such as those described herein for producing substrates having outwardly projecting needles are very suitable and economical.
Methods have been devised previously for the synthesis of microtubules. See Martin et al., "Template synthesis of organic microtubules", J. Am. Chem. Soc. 112:8976-8977 (1990); Martin, "Template synthesis of polymeric and metal microtubules", Adv. Mater. 3:457-459 (1991); and Brumlik and Martin, "Template synthesis of metal microtubules"J. Am. Chem. Soc. 113:3174-3175 (1991). In these papers, the authors describe what they call the "template synthesis" of organic or metal microtubules (or fibers) using filter membranes as molds. The organic tubules are created with the polymerization of pyrrole within a Nuclepore brand polycarbonate membrane by placing monomer on one side of the membrane and a polymerization reagent on the other. The synthesis of organic tubules can also be done electrochemically within the membrane. The membrane is removed by dissolution with dichloromethane to yield free monodisperse microtubules. Metal microtubules are generated by electrochemically depositing gold onto the surface of microporous alumina membranes, followed by dissolution of the alumina. Using this technology, dense arrays of upright metal microcapillaries have been generated that are attached to a gold film at their base. Appropriate chemistry was used such that "anchor" molecules were obtained in the membrane pores to promote generation of tubules rather than solid fibers.
U.S. Pat. No. 2,629,907 describes a method for coating models (made of wood, rubber, etc.) with a thin layer of nylon to improve the adhesion of sprayed-on metal in the manufacture of metal molds. The metal layer is removed by simple mechanical separation; solvent can be applied through a porous model to soften the nylon coating to aid removal of the metal mold. Such technology is intended for large-scale (macroscopic) molds for the manufacture of various items and would not be suitable for manufacture of substrates having microcapillaries or needles.
U.S. Pat. No. 3,996,991 also describes a method for manufacturing molds, in this case by an "investment casting" method. A model of the desired casting is made from a "thermally fusible" material, an investment (envelope) of a refractory material is made around the model, and then the thermally fusible model is removed by putting it in contact with a vaporized organic solvent. The inventors describe their technology as being particularly well suited to the production of very large castings, such as ship propellers.
U.S. Pat. No. 4,481,999 describes a method for forming thin unbacked metal foils (less than 2 microns in thickness). These foils are made by generating a soluble polymeric film (partially hydrolyzed polyvinyl alcohol) of a desired shape and then using vacuum vapor deposition to deposit a thin film of metal. The very thin metal films they generate have no special microscopic surface structure.
U.S. Pat. No. 4,867,223 describes a complex method of forming a metallic sheet with a concave-convex profile. Metal is deposited by chemical vapor deposition onto a drum with a surface of protrusions; a transparent mask and a light beam are used to control the deposition of the metal, and the metal film is physically peeled off from the drum. This technology requires sophisticated masking technology, and is designed around the fabrication of specific surface structures that might be used in a dry shaving device.
U.S. Pat. No. 4,898,623 describes an invention in which metal alloys are formed by making a plastic (polyacrylate) mold onto which metal is deposited by low-temperature arc vapor deposition. The metal layer is separated (presumably by simple physical means) from the plastic substrate and subjected to heat treatment to increase its density. This allows the forming of thin shapes from alloys that are difficult to shape by standard rolling and pressing methods.
U.S. Pat. No. 4,926,056 describes an alternate fabrication method for a microelectronic field ionizer. These highly specialized "volcano" structures are used to ionize a gas stream. The structures are fabricated by the same technology used to manufacture integrated circuits, and the described techniques are quite specific for the geometry and fabrication of field ionizers.
There has not heretofore been provided an expeditious method for manufacturing a self-supporting substrate having a plurality of spaced-apart microscopic needles projecting outwardly from one major surface of the substrate.